A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite’s resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.


Supplementary Table 1. Susceptibility of the P. falciparum lines generated through in vitro evolution in this study
and their parents to antiplasmodial compounds. The IC 50 values (mean ± SEM) for growth inhibition for the parasite lines and compounds indicated are shown, with the number of independent experiments (performed on different days) shown in brackets. Within each parasite set, all lines were tested in parallel in each experiment. For each compound, the IC 50 values for each cipargamin-resistant line were compared with those of its direct parent (Dd2 for Dd2-PfATP4 T418N,P990R ; Dd2-PfATP4 T418N,P990R for HCR1 and HCR2; and Dd2-Pol for Dd2-Pol-PfATP4 G358S ) using twotailed paired t-tests. P values ≤ 0.05 indicate statistical significance and are shown in bold. Source data are provided as a Source Data file. 1.43 ± 0.20 (7) 1420 ± 100 (7) P = 7×10 -6 (+)-SJ733 60 ± 2 (5) 331 ± 11 (5) P = 9×10 -6 21500 ± 2400 (5)  Supplementary Table 4. High confidence, non-synonymous and synonymous SNPs in coding regions and their allele frequencies called from whole-genome sequencing analyses for each of the pfatp4 gene-edited control and G358S mutant parasite lines compared to the NF54 WT parent strain. The G358S mutation in PfATP4 associated with resistance to cipargamin and (+)-SJ733 was found exclusively in the NF54 G358S-1 and NF54 G358S-2 mutant lines, in addition to the 17 silent binding-site mutations at the guide RNA cleavage site within the pfatp4 locus that were also introduced in the pfatp4 gene-edited control line (NF54 CTL ).

GENE NAME AMINO ACID CHANGE CODON CHANGE
Gene-edited NF54 parasite lines   denotes SNPs found in the edited NF54 parasite lines; mutant allele frequencies are indicated within square brackets.  Binned counts were filtered by min-mappability=50%, and a blacklist of centromere and telomere regions, then a loess fit used to correct for GC and mappability, and counts converted to copy numbers. They were inspected as copy numbers and as scaled relative to the parent strain.

Supplementary
Dd2-PfATP4 T418N,P990R and Dd2 both had the same amplification on chromosome 5. Scaled, relative copy numbers showed no features of interest. HCR1 and HCR2 copy numbers scaled relative to Dd2-PfATP4 T418N,P990R both showed a duplication on chromosome 12, and HCR2 had a relative deletion on chromosome 5, showing a reduction in the observed amplification ( Supplementary Fig. 1).
Structural variation analysis used GRIDSS, with candidate SVs inspected in Integrated Genome Viewer (IGV), found no new structural variations in Dd2-PfATP4 T418N,P990R compared to Dd2. There was a clear event in chromosome 12 in both HCR1 and HCR2 (Supplementary Fig. 1). This is a duplication from 520kb -556kb, covering nine genes including PF3D7_1211900, which is pfatp4. The breakends are in homopolymer runs of A, at Pf3D7_12_v3:520014..520039 and Pf3D7_12_v3:556464..556489.
Single-nucleotide changes and small insertions and deletions were called with SNVer. VarScan was also run but did not give usable results. All calls which also appeared in the parent strain were discarded, and remaining calls filtered by depth at least 10, alternate-frequency > 0.4, discard calls in the first and last 10% of each chromosome. When calls are filtered for 'in a coding sequence', ignore genes described as PfEMP1, rifin, stevor or pseudogene. This left 100s of SNPs and 10s of indels. On inspection most of these events were clustered in areas of low coverage, or highly repetitive regions, and some of the remainder were synonymous changes.  Immunofluorescence assays of TgATP4 WT -HA (b) and TgATP4 G419S -HA expressing parasite clones (c-e) probed with anti-HA antibodies (green) and anti-TgP30 antibodies as a marker for the plasma membrane (red). Scale bars are 2 µm. DIC, differential interference contrast transmission images. The data are from a single experiment. Source data are provided as a Source Data file.  Fig. 9. Gating strategy for flow cytometry-based quantification of Plasmodium falciparum NF54 asexual blood stage parasite proliferation in drug susceptibility assays. Intra-erythrocytic parasites were cultured at 0.3% parasitemia and 1% hematocrit for 72 hours in the presence of a range of drug concentrations that had been 2-fold serially diluted in duplicates along with drug-free controls. All assays were performed in culture media containing 10% O+ human serum. Cells were then labeled with 1X SYBR Green I (Invitrogen) and 200 nM MitoTracker Deep Red FM (Invitrogen) as nuclear stain and vital dyes, respectively. Parasite survival was assessed by flow cytometry on an Intellicyt iQue3 (Essen Bioscience). Between 9,000 and 15,000 events were counted per sample. Data show a control gating of untreated parasite cultures from a representative dose-response assay. a. Forward (FSC-H) and side scatter (SSC-H) gating of the total red blood cell (RBC) population, representing 97.0% of the total events counted. b. Gating strategy used to quantify viable intracellular parasites, which are positively stained by both MitoTracker Deep Red and SYBR Green I and appear as events in the upper right gate (live parasites, 3.80% parasitemia). Uninfected erythrocytes appear in the lower left gate as MitoTracker Deep Red and SYBR Green I negative populations. The gating strategy shown here was used to generate the data shown in Fig. 2c